Christina Cheers

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Abstract Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1+ Ly-1+2+ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia+ B lymphocytes were not required, and Ia+ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1+2+ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.

Resident and Monocyte-Derived Dendritic Cells Become Dominant IL-12 Producers under Different Conditions and Signaling Pathways

IL-12 is such a pivotal cytokine that it has been called the third signal for T cell activation, TCR engagement being the first and costimulation being the second. It has been generally viewed that the resident CD8+ dendritic cell (DC) subset is the predominant IL-12–producing cell type. In this study, we found, although this is so under steady state conditions, under inflammatory conditions monocyte-derived DC (mDC) became a major cell type producing IL-12. Depletion of either type of DC resulted in reduced production of IL-12 in vivo. For CD8+ DC, IL-12 production could be stimulated by various pathways viz. signaling through MyD88, Trif, or nucleotide-binding oligomerization domain (Nod)-like receptors. In contrast, for mDC, IL-12 production was mainly dependent on MyD88 signaling. Thus, conventional DCs and mDCs use different pathways to regulate IL-12 production.

Restriction in adoptive transfer of resistance to Listeria monocytogenes. II. Use of congenic and mutant mice show transfer to be H-2K restricted.

Adoptive transfer of cell-mediated immunity to the facultative intracellular bacterium Listeria monocytogenes is restricted by the H-2 complex of mice. Using C57BL/10 and C57BL/6 congenic strains of mice it was shown that compatibility of the H-2K locus, not the I region, was essential and sufficient for adoptive transfer and that H-2D compatibility was not relevant. Mutation at the H-2K locus prevented adoptive transfer, while mutation at the Ia-1 locus, as in the B6.C-H-2bm12 mutant of C57BL/6, did not affect adoptive transfer. The contrast between these findings and the previously accepted I region restriction of adoptive transfer of Listeria immunity is discussed.

Bystander Activation of CD8+ T Lymphocytes during Experimental Mycobacterial Infection

ABSTRACT Infection of C57BL/6 mice with Mycobacterium avium leads to the activation of both CD4+ and CD8+ gamma interferon (IFN-γ)-producing T cells, although the CD8+ cells play no role in protection against infection. Using transfer of different lines of transgenic T cells with T-cell receptors (TCRs) which recognize irrelevant antigens, we show here that transferred CD8+ T cells from two of the three lines were activated to the same degree as the host cells, suggesting that the majority of the IFN-γ-producing CD8+ T cells of the host represented bystander activation. The third line, specific for the male HY antigen, showed no activation. Activation required the participation of the CD28 coreceptor on T cells and was unaffected by the removal of CD44hi (memory phenotype) T cells. The transferred CD8+ T cells proliferated in vivo, although this was not essential for IFN-γ production. Taken together, these data are highly reminiscent of homeostatic proliferation of TCR transgenic T cells upon transfer to lymphopenic hosts, and suggest low-affinity stimulation through the TCR, possibly by self peptides. The findings are discussed in relation to homeostatic proliferation and their significance in the possible induction of autoimmune disease.

Macrophage activation during experimental murine brucellosis: II. Inhibition of in vitro lymphocyte proliferation by brucella-activated macrophages

Abstract During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.

Oxidised mannan as a novel adjuvant inducing mucosal IgA production

Mannan, oxidatively coupled to recombinant protein antigens, has here been tested as a possible adjuvant for the production of antibody on the mucosa. Given intranasally, but not intraperitoneally, mannan markedly enhanced the production of IgA, IgG1 and IgG2a in the serum, and IgA locally in the lung and at remote mucosal sites, including tears, vaginal and salivary secretions. Oxidative coupling was critical to its action, since neither mannan simply mixed with protein nor mannan-protein conjugates which had been reduced by treatment with sodium borohydride, acted as adjuvants. Oxidatively coupled mannan was compared with the widely studied mucosal adjuvant, cholera toxin (CT). The use of oxidised mannan as an adjuvant induced better responses than CT judged by the induction of IgA in serum, vaginal washings and saliva. Thus, oxidised mannan, which is non-toxic and can be administered without injection, is a suitable adjuvant coupled with protective antigens for vaccinating against a number of infections that occur via the mucous membranes.

Functional deficiencies of peritoneal cells from gene-targeted mice lacking G-CSF or GM-CSF.

Gene‐targeted mice lacking the hemopoietic growth factors, granulocyte colony‐stimulating factor (G‐CSF) or granulocyte‐macrophage (GM)‐CSF, show increased susceptibility to infection with the facultative intracellular bacterium, Listeria monocytogenes. The resident peritoneal cell populations from G‐CSF‐/‐ and GM–CSF‐/‐ mice showed reduced production of the bactericidal molecule nitric oxide. Macrophage‐mediated tumoricidal activity and phagocytosis of Listeria were reduced in G‐CSF‐/‐, but not in GM‐CSF‐/‐, mice. In G‐CSF‐/‐ mice, there was an unexpected expansion (from 18% in WT to 38%) of a population of cells with morphology intermediate between typical macrophages and typical lymphocytes. These cells had some of the features of poorly differentiated macrophages, being adherent to plastic but poorly phagocytic, nonspecific esterase positive but myeloperoxidase negative. They were largely negative for the macrophage marker F4/80 and for Thy1, B220, and Gr1. Their disproportionate presence, and the corresponding deficiency in typical macrophages, possibly accounts for some of the functional deficiencies observed in G‐CSF‐/‐ mice. J. Leukoc. Biol. 65: 256–264; 1999.

T‐cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection

Listeria monocytogenes infection of mice leads to a rapid expansion of activated T cells, followed by a decline in specific cells once the bacteria are eliminated. In order to define the relationship between T‐cell proliferation and activation, and to investigate the role of apoptosis in limiting the expansion, the expression of activation markers, uptake of 5‐bromo‐2′‐deoxyuridine (BrdU) in vivo and the incidence of apoptosis was investigated. Increased numbers of T cells expressing the activated phenotype CD25+, CD44hi and CD62Llo were detected 4 days after infection. Expression of CD25 (IL‐2Rα chain) on CD4+ and CD8+ T cells peaked at this time and returned to normal by day 7. In contrast, CD44hi and CD62Llo persisted, with the maximum proportion occurring at 7 days after infection. This was accompanied by a burst of in vivo proliferation of CD4+ and CD8+ T cells occurring between day 5 and 7. Apoptosis, which is presumably needed to control this expansion of T cells, also peaked at 7 days after infection. Apoptosis occurred preferentially amongst T cells which had proliferated. Most but not all proliferating T cells had down‐regulated their CD62L marker. While most apoptotic T cells were CD62Llo, again not all had down‐regulated this marker. Hence, CD25 expression peaked early, but expression of other activation markers, in vivo proliferation and apoptosis coincided after Listeria infection. T cells that had proliferated were over‐represented in the apoptotic population.

Macrophage activation during experimental murine brucellosis: III. Do macrophages exert feedback control during brucellosis?

Abstract The role played by macrophages in feedback inhibition of the immune response during murine brucellosis, allowing establishment of chronic infection, was investigated using a number of approaches. First, it was shown that the degree of splenomegaly (a measure of macrophage influx) following infection with Brucella abortus strain 19 did not correlate with the course of bacterial numbers in the spleen of CBA, BALB/c, and C57B1/10 mice. Second, it was shown that a rough, mucoid mutant, B. abortus strain 19R, although causing a chronic infection, did not induce splenomegaly. Nor could “suppressor macrophages” be demonstrated in these spleens. Delayed-type hypersensitivity during this infection was lower than with B. abortus strain 19. Third, no nonspecific suppression of unrelated immune responses in CBA mice infected with B. abortus strain 19 could be demonstrated, despite the very large numbers of macrophages in the spleen. The responses tested included delayed-type hypersensitivity and cell-mediated resistance to Listeria monocytogenes , antibody response to sheep erythrocytes (both serum antibody and plaque-forming cells in the spleen) and skin-graft rejection.

Use of recombinant viruses to deliver cytokines influencing the course of experimental bacterial infection

The feasibility of using viral constructs expressing cytokine genes to influence the course of bacterial infection was tested in mice. The mice were first infected with vaccinia or fowlpox viruses expressing the cytokine of interest, then challenged with the facultative intracellular bacterial pathogen Listeria monocytogenes. The course of infection was assessed by subsequent bacterial counts. Expression of IFN‐γ or TNF was protective. Vaccinia virus was more efficient at delivering IFN‐γ‐mediated protection than was fowlpox virus, which is unable to proliferate in mammalian cells. The effect of vaccinia‐IFN‐γ was more apparent in the liver, where vaccinia proliferates to high titres (> 109), than in the spleen, where only 103 vaccinia were isolated. Vaccinia virus expressing IL‐4 exacerbated infection. Interleukin‐4 exacerbation was T cell independent and was reflected in the failure of macrophage activation, possibly due to suppression of NK cells, which are a source of IFN‐γ early in infection. The clear indication of protection by some cytokines in this prophylactic model appears to justify further study of the therapeutic effects of cytokine‐expressing viruses in chronic bacterial infections, especially where a cytokine defect is suspected.